*Target (protein/gene name): Glucose-1-phosphatase *NCBI Gene # or RefSeq#: 1247561 *Protein ID (NP or XP #) or Wolbachia#: NP_455612.1 *Organism (including strain): Salmonella Enterica (str. 08-0047) Etiologic Risk Group (see link below): Category B Priority Pathogens */Disease Information (sort of like the Intro to your Mini Research Write up): Salmonella is among the most common food-borne diseases, causing approximately 380 deaths per year in the US. Those with particularly vulnerable immune systems like children and the elderly are most prone to this disease. Salmonella enterica is one of the two most common strains of salmonella, along with S. Typhimurium. Link to TDR Targets page (if present): Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: Used in glycolysis and gluconeogenesis pathways, essential for energy synthesis. Important in initial pathway for glycolysis, however, alternate routes available. "The presence of a hexose phosphatase with similar relative specificity in the periplasmic space of Salmonella typhimurium has been reported (25). The ability of E. coli strains lacking the hexose phosphate transport system to utilize glucose-i-phosphate (G-1-P) as the sole carbon source has also been considered as an indication that some periplasmic phosphatase could efficiently cleave G-1-P (13). Inactivating agp on the chromosome of a wild-type strain indicated that GlPase was indeed absolutely required for the growth of E. coli in a high-phosphate medium containing G-1-P as the sole carbon source" [paper]
Complex of proteins?: No Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Inhibitors found: Phosphate (product inhibition) [3,4]; F- [4], Arsenate [4] [paper] *EC#: 3.1.3.10 Link to BRENDA EC# page: --BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog # Enzyme assay with pNPP as substrate, with potassium phalate buffer (E. coli) [paper]
"Glucose-1-phosphatase assays were performed in 200 ml of 0.1 M sodium acetate buffer pH 4.5 using 5 mM of glucose-1- phosphate as the substrate at 310 K for 10 min. The reaction was stopped by adding 133 ml of a mixture of ammonium molybdate, ammonium vanadate and nitric acid (Engelen et al., 1994). The concentration of inorganic phosphate released was determined by comparison with a K2HPO4 standard measured at 405 nm." [paper]
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 1NT4 -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: 94% ---- Max % Identities:83% ---- % Positives: 90% ---- Chain used for homology: QTVPEGYQLQQVLMMSRANLRAPLANNGSVLEQSTPNKWPEWDVPGGQLTTKGGVLEVYMGHYMREWLAE
Current Inhibitors: Phosphate (product inhibition) [3,4]; F- [4], Arsenate [4]
Expression Information (has it been expressed in bacterial cells): Structural gene AGP can be cloned to overexpress E. coli phosphatases, including Glucose-1-phosphatase. [paper, paper] Purification Method: "Glucose-1-phosphatase was purified from the osmotic shock fluid of a 1-liter culture of strain SBS1405(pEP1376) grown to early stationary phase in TYE medium plus ampicillin. Low-molecular-weight proteins were removed, and the enzyme was concentrated with an Amicon concentrator equipped with an UM20 membrane. The concentrated preparation was chromatographed on a fast protein liquid chromatography Mono-Q column (Pharmacia) in 5 mM Tris hydrochloride buffer (pH 7.8) with a linear gradient of NaCl (0 to 500 mM)." [paper] Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
NANRDLMKIEYVYQSARQLRNAEALTLKSPAQRVTLELKGCPVDANGFCPLDKFDNVMNTAAK *length of your protein in Amino Acids: 413 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 45629.85 kD Molar Extinction coefficient of your protein at 280 nm wavelength: 56840 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only): *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 1247561
*Protein ID (NP or XP #) or Wolbachia#: NP_455612.1
*Organism (including strain): Salmonella Enterica (str. 08-0047)
Etiologic Risk Group (see link below): Category B Priority Pathogens
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Salmonella is among the most common food-borne diseases, causing approximately 380 deaths per year in the US. Those with particularly vulnerable immune systems like children and the elderly are most prone to this disease. Salmonella enterica is one of the two most common strains of salmonella, along with S. Typhimurium.
Link to TDR Targets page (if present):
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: Used in glycolysis and gluconeogenesis pathways, essential for energy synthesis. Important in initial pathway for glycolysis, however, alternate routes available.
"The presence of a hexose phosphatase with similar relative specificity in the periplasmic space of Salmonella typhimurium has been reported (25). The ability of E. coli strains lacking the hexose phosphate transport system to utilize glucose-i-phosphate (G-1-P) as the sole carbon source has also been considered as an indication that some periplasmic phosphatase could efficiently cleave G-1-P (13). Inactivating agp on the chromosome of a wild-type strain indicated that GlPase was indeed absolutely required for the growth of E. coli in a high-phosphate medium containing G-1-P as the sole carbon source" [paper]
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Two amino acid chains and two ligands in ASU. Likely a multimer.
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Inhibitors found: Phosphate (product inhibition) [3,4]; F- [4], Arsenate [4] [paper]
*EC#: 3.1.3.10
Link to BRENDA EC# page:
--BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Enzyme assay with pNPP as substrate, with potassium phalate buffer (E. coli) [paper]
"Glucose-1-phosphatase assays were performed in 200 ml of 0.1 M sodium acetate buffer pH 4.5 using 5 mM of glucose-1- phosphate as the substrate at 310 K for 10 min. The reaction was stopped by adding 133 ml of a mixture of ammonium molybdate, ammonium vanadate and nitric acid (Engelen et al., 1994). The concentration of inorganic phosphate released was determined by comparison with a K2HPO4 standard measured at 405 nm." [paper]
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1NT4
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 94%
---- Max % Identities:83%
---- % Positives: 90%
---- Chain used for homology:
QTVPEGYQLQQVLMMSRANLRAPLANNGSVLEQSTPNKWPEWDVPGGQLTTKGGVLEVYMGHYMREWLAE
QGMVKSGECPPPYTVYAYANSLQRTVATAQFFITGAFPGCDIPVHHQEKMGTMDPTFNPVITDDSAAFSE
QAVAAMEKELSKLQLTDSYQLLEKIVNYKDSPACKEKQQCSLVDGKNTFSAKYQQEPGVSGPLKVGNSLV
DAFTLQYYEGFPMDQVAWGEIKSDQQWKVLSKLKNGYQDSLFTSPEVARNVAKPLVSYIDKALVTDRTSA
PKITVLVGHDSNIASLLTALDFKPYQLHDQNERTPIGGKIVFQRWHDSKANRDLMKIEYVYQSAEQLRNA
DALTLQAPAQRVTLELSGCPIDADGFCPMDKFDSVLNEAVK
Current Inhibitors:
Phosphate (product inhibition) [3,4]; F- [4], Arsenate [4]
Expression Information (has it been expressed in bacterial cells):
Structural gene AGP can be cloned to overexpress E. coli phosphatases, including Glucose-1-phosphatase. [paper, paper]
Purification Method:
"Glucose-1-phosphatase was purified from the osmotic shock fluid of a 1-liter culture of strain SBS1405(pEP1376) grown to early stationary phase in TYE medium plus ampicillin. Low-molecular-weight proteins were removed, and the enzyme was concentrated with an Amicon concentrator equipped with an UM20 membrane. The concentrated preparation was chromatographed on a fast protein liquid chromatography Mono-Q column (Pharmacia) in 5 mM Tris hydrochloride buffer (pH 7.8) with a linear gradient of NaCl (0 to 500 mM)." [paper]
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MKKSLLAVAVAGAVLLSSAVQAQTTPEGYQLQQVLMMSRHNLRAPLANNGNVLAQSTPNAWPAWDVPGGQ
LTTKGGVLEVYMGHYTREWLVAQGLIPSGECPAPDTVYAYANSLQRTVATAQFFITSAFPGCDIPVHHQE
KMGTMDPTFNPVITDDSAAFRQQAVQAMEKARSQLHLDESYKLLEQITHYQDSPSCKEKHQCSLIDAKDT
FSANYQQEPGVQGPLKVGNSLVDAFTLQYYEGFPMDQVAWGGIHTDRQWKVLSKLKNGYQDSLFTSPTVA
RNVAAPLVKYIDKVLVAERVSAPKVTVLVGHDSNIASLLTALDFKPYQLHDQYERTPIGGQLVFQRWHDG
NANRDLMKIEYVYQSARQLRNAEALTLKSPAQRVTLELKGCPVDANGFCPLDKFDNVMNTAAK
*length of your protein in Amino Acids: 413
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 45629.85 kD
Molar Extinction coefficient of your protein at 280 nm wavelength: 56840
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**